Abstract DNA Double strand breaks (DSBs) pose a serious threat to genomic integrity and cell survival, and are drivers of cancer. Non-homologous end joining (NHEJ) is responsible for repairing the majority of these breaks in higher eukaryotes. The NHEJ synaptic complex consists of the core factors Ku, DNA-PKcs, XRCC4/LigIV, and XLF. Together these repair proteins must first recognize the DSB, tether the DNA ends together, and then process and align them for direct ligation by the XRCC4/LigIV complex. By utilizing single-molecule FRET (smFRET) to monitor DNA end synapsis in real time within the context of the physiological Xenopus egg extract system, our lab has recently shown that repair by NHEJ proceeds through at least two distinct stages. DNA ends are first synapsed in a Long Range Complex where the ends are more than 10 nm apart. Only Ku and DNA- PKcs are required to form this state. Next, the DNA ends are closely aligned prior to ligation in a Short Range Complex. The transition to the Short Range Complex requires DNA-PKcs kinase activity and the presence of XLF and XRCC4/LigIV. However, LigIV?s catalytic activity is not required to form the Short Range Complex. What drives the transition between these two distinct states remains unclear. In this proposal, I aim to determine the role of XRCC4/LigIV in DNA end synapsis through continued use of the Xenopus egg extract system in single-molecule fluorescence experiments. Building on preliminary data demonstrating that a single copy of XLF is sufficient for end joining, I will determine the number of XRCC4/LigIV and free XRCC4 present and acting at a double strand breaks. I will generate labeled XRCC4/LigIV and free XRCC4 constructs to directly determine the copy number of each at DSBs using 3-color single-molecule imaging. Additionally, I will determine whether interaction with XLF is required to retain or stabilize XRCC4/LigIV or free XRCC4 within the Short Range Complex. The observation that LigIV, but not its catalytic activity, is needed to progress to the Short Range Complex suggests that LigIV has a structural role in synaptic complex assembly. To determine the basis of this role, I will generate a series of N-terminal truncation mutants, and reveal the minimal LigIV domain requirements for Short Range Complex formation. Whether the interactions that drive Short Range Complex formation involve DNA or are required for LigIV to gain access to the DNA ends is unclear. I will employ a 3-color smFRET strategy to measure when LigIV interacts with the DNA ends relative to the formation of the Short Range Complex. These findings will have the potential for significant impact in the field, as the role(s) of the most critical component of NHEJ, XRCC4/LigIV, remains poorly defined. Elucidating the mechanism of XRCC4/LigIV, and more broadly NHEJ, will allow for a better understanding of disease and inform the development of new therapies and biotechnology applications.